Coating and Corruption of Human Neutrophils by Bacterial Outer Membrane Vesicles

ABSTRACT Porphyromonas gingivalis is a keystone oral pathogen that successfully manipulates the human innate immune defenses, resulting in a chronic proinflammatory state of periodontal tissues and beyond. Here, we demonstrate that secreted outer membrane vesicles (OMVs) are deployed by P. gingivalis to selectively coat and activate human neutrophils, thereby provoking degranulation without neutrophil killing. Secreted granule components with antibacterial activity, especially LL-37 and myeloperoxidase (MPO), are subsequently degraded by potent OMV-bound proteases known as gingipains, thereby ensuring bacterial survival. In contrast to neutrophils, the P. gingivalis OMVs are efficiently internalized by macrophages and epithelial cells. Importantly, we show that neutrophil coating is a conserved feature displayed by OMVs of at least one other oral pathogen, namely, Aggregatibacter actinomycetemcomitans. We conclude that P. gingivalis deploys its OMVs for a neutrophil-deceptive strategy to create a favorable inflammatory niche and escape killing. IMPORTANCE Severe periodontitis is a dysbiotic inflammatory disease that affects about 15% of the adult population, making it one of the most prevalent diseases worldwide. Importantly, periodontitis has been associated with the development of nonoral diseases, such as rheumatoid arthritis, pancreatic cancer, and Alzheimer’s disease. Periodontal pathogens implicated in periodontitis can survive in the oral cavity only by avoiding the insults of neutrophils while at the same time promoting an inflamed environment where they successfully thrive. Our present findings show that outer membrane vesicles secreted by the keystone pathogen Porphyromonas gingivalis provide an effective delivery tool of virulence factors that protect the bacterium from being killed while simultaneously activating human neutrophils.


Supplementary Figure S1
Supplementary Figure S1. OMVs (1 µg) of P. gingivalis coat the neutrophil and are trapped in NETs. (A and B) Confocal fluorescence microscopy images of human neutrophils challenged with OMVs from P. gingivalis W83 or W83ΔPPAD. (A) an amount of 1 µg of OMVs was used to detect single OMVfluorescent signals. (B) NETs trapping OMVs of P. gingivalis. DAPI was used to stain the neutrophils' nuclei (blue) and OMVs were labelled with P. gingivalis-specific polyclonal rabbit antibodies and secondary goat-anti-rabbit antibodies labelled with AlexaFluor488 (green). Scale bars mark 20 µm. Figure S2. Neutrophils do not internalize OMVs of P. gingivalis within 150 minutes. (A and B) Confocal fluorescent microscopic images of neutrophils at different time points (30, 90 and 150 min) after addition of 5 µg of W83 OMVs (A) or W83ΔPPAD OMVs (B). DAPI was used to stain the neutrophils' nuclei (blue) and OMVs were labelled using P. gingivalis-specific polyclonal rabbit antibodies and secondary goat-anti-rabbit antibodies labelled with AlexaFluor488 (green). Scale bars in the panels with the merged images mark 25 or 100 µm. Figure S3. Neutrophil, macrophages and A253 cells + OMVs. (A, B and C) Representative confocal fluorescence microscopy images of neutrophils (A), macrophages (B) and A253 cells (C) challenged with 5 µg of OMVs of P. gingivalis W83 or W83ΔPPAD, corresponding to Figure 2 in the main manuscript. DAPI was used to stain the cells' nuclei (blue) and Phalloidin-TRITC (red) was used to stain actin. Additionally, OMVs were labelled with P. gingivalis-specific polyclonal rabbit antibodies and secondary goat-anti-rabbit antibodies labelled with AlexaFluor488 (green). Scale bars in the panels with the merged images mark 50 µm.

Supplementary Figure S4
Supplementary Figure S4. OMVs of P. gingivalis bind to the neutrophil's surface independently of gingipain activity or the presence of plasma. (A and B) Confocal fluorescence microscopy images of neutrophils after addition of 5 µg of OMVs isolated from P. gingivalis W83 or W83ΔPPAD in the presence of gingipain inhibitors (A) or the absence of human plasma (B). DAPI was used to stain the neutrophils' nuclei (blue) and Phalloidin-TRITC was used to stain actin (red). Additionally, OMVs were labelled with P. gingivalis-specific polyclonal rabbit antibodies and secondary goat-anti-rabbit antibodies labelled with AlexaFluor488 (green). Scale bars in the panels with the merged images mark 50 µm. (A) Representative confocal microscopy images of 5 µg of OMVs isolated from P. gingivalis W83 or W83ΔPPAD trapped in formed NETs. DAPI was used to stain the neutrophils' nuclei and NETs (blue) and Phalloidin-TRITC was used to stain actin (red). Additionally, OMVs were labelled with P. gingivalisspecific polyclonal rabbit antibodies and secondary goat-anti-rabbit antibodies labelled with AlexaFluor488 (green). Unchallenged neutrophils were used as a control (C-). Scale bars in the panels with the merged images mark 50 µm. The images in this Figure  Representative confocal microscopy images of neutrophils challenged with P. gingivalis strains W83, W83ΔPPAD, ATCC 33277 or ATCCΔPPAD, the clinical isolate P. gingivalis #6, or A. actinomycetemcomitans 30R. DAPI was used to stain the neutrophils' nuclei or extracellular DNA (blue) and Phalloidin-TRITC was used to stain actin (red). Bacterial cells were identified with P. gingivalis-or A. actinomycetemcomitans-specific polyclonal rabbit antibodies and secondary goatanti-rabbit antibodies labelled with AlexaFluor488 (green). Scale bars in the panels with the merged images mark 20 µm. The images in this Figure correspond to Figure 7 in the main manuscript.
Supplementary Video S1. Neutrophils challenged with OMVs of P. gingivalis W83. Threedimensional reconstructions from Z-stacks of two-dimensional confocal microscopy images of neutrophils challenged with 5 µg of OMVs of P. gingivalis W83. DAPI was used to stain the neutrophils' nuclei (blue). Bacterial OMVs were identified with P. gingivalis-specific polyclonal rabbit antibodies and secondary goat-anti-rabbit antibodies labelled with AlexaFluor488 (green).
Supplementary Video S2. Neutrophils challenged with OMVs of P. gingivalis W83ΔPPAD. Threedimensional reconstructions from Z-stacks of two-dimensional confocal microscopy images of neutrophils challenged with 5 µg of OMVs of P. gingivalis W83ΔPPAD. DAPI was used to stain the neutrophils' nuclei (blue). Bacterial OMVs were identified with P. gingivalis-specific polyclonal rabbit antibodies and secondary goat-anti-rabbit antibodies labelled with AlexaFluor488 (green).

Supplementary Video S3. A253 epithelial cells challenged with OMVs of P. gingivalis W83.
Three-dimensional reconstructions from Z-stacks of two-dimensional confocal microscopy images of A253 epithelial cells challenged with OMVs of P. gingivalis W83. DAPI was used to stain the epithelial cells' nuclei (blue) and Phalloidin-TRITC was used to stain actin (red). Additionally, OMVs were labelled with P. gingivalis-specific polyclonal rabbit antibodies and secondary goat-anti-rabbit antibodies labelled with AlexaFluor488 (green). Note that the Videos 3A and 3B present different angles of the same video.

Supplementary Video S4. A253 epithelial cells challenged with OMVs of P. gingivalis W83ΔPPAD.
Three-dimensional reconstructions from Z-stacks of two-dimensional confocal microscopy images of A253 epithelial cells challenged with OMVs P. gingivalis W83ΔPPAD. DAPI was used to stain the epithelial cells' nuclei (blue) and Phalloidin-TRITC was used to stain actin (red). Additionally, OMVs were labelled with P. gingivalis-specific polyclonal rabbit antibodies and secondary goat-anti-rabbit antibodies labelled with AlexaFluor488 (green). Note that the Videos 4A and 4B present different angles of the same video.
Supplementary Table S1. Mass spectrometry analysis of a granule-derived MPO preparation incubated with or without recombinant PPAD. Granule-derived MPO was incubated overnight at 37 o C with or without recombinant PPAD. Subsequently, the samples were analyzed by MS. The determined protein LFQ intensities show that MPO was the most abundant protein in the samples and that MPO was identified in all samples. PPAD was only identified with two unique peptides in the 3rd replicate sample. A potential citrullination site was detected on arginine 569 of MPO and manually validated.